Combination of pilocarpin and methimazol for treating charcot-marietooth disease and related disorders

ABSTRACT

The present invention relates to compositions and methods for the treatment of the Charcot-Marie-Tooth disease and related peripheral neuropathies. More particularly, the invention relates to combined therapies for treating said disease by affecting simultaneously muscarinic receptor signalling and thyroid hormone pathway in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/999,125, filed Aug. 3, 2011, which is the U.S. national stageapplication of International Patent Application No. PCT/EP2009/057544,filed Jun. 17, 2009.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Dec. 14, 2010 and is 13 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to compositions and methods for thetreatment of the Charcot-Marie-Tooth disease and related disorders.

Charcot-Marie-Tooth disease (“CMT”) is an orphan genetic peripheral polyneuropathy. Affecting approximately 1 in 2,500 individuals, this diseaseis the most common inherited disorder of the peripheral nervous system.Its onset typically occurs during the first or second decade of life,although it may be detected in infancy. Course of disease is chronicwith gradual neuromuscular degeneration. The disease is invalidatingwith cases of accompanying neurological pain and extreme musculardisability. CMT is one of the best studied genetic pathologies withapproximately 30,000 cases in France. While a majority of CMT patientsharbour a duplication of a chromosome 17 fragment containing a myelingene: PMP22 (form CMT1A), two dozens of genes have been implicated indifferent forms of CMT. Accordingly, although monogenic in origin, thispathology manifests clinical heterogeneity due to possible modulatorgenes. The genes mutated in CMT patients are clustering around tightlyconnected molecular pathways affecting differentiation of Schwann cellsor neurons or changing interplay of these cells in peripheral nerves.

PMP22 is a major component of myelin expressed in the compact portion ofessentially all myelinated fibers in the peripheral nervous system andis produced predominantly by Schwann cells. Furthermore, PMP22 gene isassumed to be involved in the development of neoplasia in patients withneurofibromatosis, an autosomal dominant disorder characterized bycafe-au-lait spots and fibromatous tumors of the skin. A modest,1.5-fold overexpression of a normal PMP22 protein is also observed inSchwann cells heterozygous for the duplication in CMT patients (in somerare cases, CMT1A-like phenotype can be also linked to structuralmutations in PMP22 protein) (Lupski et al., 1992; Suter et al., 1992;Roa et al., 1993; Thomas et al., 1997; Suter & Scherer, 2003; Nave &Sereda, 2007). Direct evidence that abnormal PMP22 gene dosage causes aCMT1A-like phenotype was provided by transgenic experiments in rodentmodels with overexpression of PMP22 protein (Niemann et al., 1999; Pereaet al., 2001; Robaglia-Schlupp et al., 2002; Meyer et al., 2006; Sereda& Nave, 2006).

Furthermore, therapeutic interventions with inhibitor of progesteronereceptor and ascorbic acid decreased this expression in the transgenicanimals ameliorating or slowing the progression of disease phenotype(Sereda et al., 2003; Passage et al., 2004; Meyer zu Horste et al.,2007).

Bassi and al., 1978, relates to the use of methimazole in the treatmentof the thyrotoxicose associated with neuropathy and encephalomyelitis.

WO 00/20024 of Celtrix concerns the use of methimazole for alleviatingsymptoms of an IGF-dependent disorder which is a thyroid disorder.

WO2004/019938 relates to the use of pilocarpine in the treatment ofsyndromes of neuropathic pain.

The use of pilocarpine, alone, for the treatment of myotonic papillaryabnormalities was suggested by Keltner et al., 1975. However, the use ofpilocarpine for treatment of CMT was not described. Moreover, the use ofpilocarpine in combination with any other compound was not suggestedeither.

In conclusion, there is a need for efficient and approved therapy fortreating CMT disease.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide new therapeuticapproaches for treating CMT and related disorders. More specifically,the inventors have identified novel combination therapies whicheffectively affect pathways leading to CMT and related disorders, andrepresent new approaches for the treatment of these disorders. Theinvention therefore provides novel combination products andcompositions, as well as the uses thereof for treating CMT disease andrelated disorders.

An object of this invention more specifically relates to the use of acombination of compounds for (the manufacture of a medicament for)treating CMT or a related disorder, wherein said combination ofcompounds is selected from a muscarinic receptor agonist or a prodrugthereof and an inhibitor of thyroid hormone synthesis or a prodrugthereof.

Another object of this invention resides in a combination productcomprising a muscarinic receptor agonist and an inhibitor of thyroidhormone synthesis, or a prodrug thereof, for a grouped or separateadministration to a subject, simultaneously or sequentially.

The invention also relates to a pharmaceutical composition comprising amuscarinic receptor agonist and an inhibitor of thyroid hormonesynthesis, or a prodrug thereof, and a pharmaceutically acceptablecarrier or excipient.

In a most preferred embodiment, the muscarinic receptor agonist is(3S,4R)-3-ethyl-4-[(3-methylimidazol-4-yl)methyl]oxolan-2-one (CAS92-13-7, which is an active agent of pilocarpine pharmaceutical), or aprodrug thereof.

Furthermore in a most preferred embodiment, the inhibitor of thyroidhormone synthesis further displays activity in prostaglandin signalling.Preferred examples of such compounds include1-methyl-3H-imidazole-2-thione (CAS 60-56-0, which is an active agent ofmethimazole pharmaceutical), or a prodrug thereof, such as carbimazole.

In another embodiment, the invention relates to a composition comprisinga muscarinic receptor agonist and an inhibitor of thyroid hormonesynthesis for the treatment of CMT in a subject, wherein the treatmentfurther comprises a step of determining whether the patient has CMT1A.

In this regard, a particular object of this invention relates to acomposition comprising pilocarpine, or a prodrug thereof, andmethimazole, or a prodrug thereof and, optionally, a pharmaceuticallyacceptable carrier or excipient. As shown in the examples, such acombination allows to effectively treat CMT in recognized animal models.

The products or compositions of this invention may further comprise atleast one additional active compound, preferentially selected from thegroup consisting of rapamycin, baclofen, sorbitol, mifepristone,naltrexone, flurbiprofen and ketoprofen.

In a preferred embodiment, the composition according to the inventioncomprises at least:

-   -   pilocarpine, methimazole and mifepristone;    -   pilocarpine, methimazole and baclofen; or    -   pilocarpine, methimazole and sorbitol.

In another preferred embodiment, the composition according to theinvention comprises at least:

-   -   pilocarpine, methimazole, mifepristone, baclofen and sorbitol;        or    -   pilocarpine, methimazole, mifepristone, baclofen, sorbitol and        naltrexone.

In other embodiments, the present invention relates to a product orcomposition comprising methimazole, pilocarpine and two additionalactive compounds, preferably mifepristone and sorbitol.

In other embodiments, the present invention relates to a product orcomposition comprising methimazole, pilocarpine and three additionalactive compounds, preferably:

-   -   mifepristone, baclofen and sorbitol; or    -   mifepristone, sorbitol and rapamycin; or    -   mifepristone, sorbitol and ketoprofen; or    -   mifepristone, sorbitol and flurbiprofen.

In other embodiments, the present invention relates to a product orcomposition comprising methimazole, pilocarpine and four additionalactive compounds, preferably:

-   -   mifepristone, sorbitol, baclofen and naltrexone;    -   mifepristone, sorbitol, baclofen and rapamycin;    -   mifepristone, sorbitol, naltrexone and rapamycin.

In other embodiments, the invention relates to a product or composition,which comprises any drug combination as disclosed in Table 1.

In other specific embodiments, the present invention relates to aproduct or composition comprising:

-   -   methimazole and baclofen; or    -   methimazole and cevimeline.

In another embodiment, the invention also relates to a product orcomposition comprising pilocarpine and propylthiouracil.

Another object of the invention relates to the use of a combination ofpilocarpine with methimazole or these two compounds alone, or incombination(s) with other compounds enhancing their effect for the(manufacture of a medicament for the) treatment of CMT or a relateddisorder.

Another object of the invention relates to the use of a combination ofpilocarpine with methimazole or these two compounds alone, or incombination(s) with other compounds enhancing their effect for the(manufacture of a medicament for the) treatment of toxic neuropathy.

Another object of the invention relates to the use of a combination ofpilocarpine with methimazole or these two compounds alone, or incombination(s) with other compounds enhancing their effect for the(manufacture of a medicament for the) treatment of ALS (AmyotrophicLateral Sclerosis).

Preferably, the compounds enhancing the effect of pilocarpine andmethimazole combination, for the treatment of CMT, are selected from thegroup consisting of mifepristone, baclofen, sorbitol, naltrexone,rapamycin, flurbiprofen and ketoprofen.

In a variant, a pilocarpine and methimazole mixture is used, for thetreatment of CMT or a related disorder, in combination with oneadditional active compound, preferably mifepristone or baclofen.

In another variant a pilocarpine and methimazole mixture is used incombination with two additional active compounds, preferablymifepristone and sorbitol.

In another variant a pilocarpine and methimazole mixture is used incombination with three additional active compounds, preferablyconsisting of mifepristone and sorbitol in combination with a thirdcompound selected from baclofen, rapamycin, ketoprofen or furbiprofen.

In another variant a pilocarpine and methimazole mixture is used incombination with four additional active compounds, preferablymifepristone, sorbitol, baclofen and naltrexone or rapamycin; ormifepristone, sorbitol, naltrexone and rapamycin.

The invention further provides a method for treating CMT or a relateddisorder, particularly CMT, comprising administering to a subject inneed thereof an effective amount of combination of a muscarinic receptoragonist, or a prodrug thereof, and an inhibitor of thyroid hormonesynthesis, or a prodrug thereof.

The invention further relates to a method of treating CMT in a subject,comprising administering to the subject an effective amount of acombination of pilocarpine, or a prodrug thereof, and methimazole, or aprodrug thereof.

The invention also relates to a method of treating CMT in a subject,comprising administering to the subject an effective amount of acombination of pilocarpine and methimazole with at least one additionalactive compound, preferentially selected from the group of rapamycin,baclofen, sorbitol, mifepristone, naltrexone, flurbiprofen andketoprofen.

In variants, the present invention also relates to a method of treatingCMT in a subject, comprising administering to the subject an effectiveamount of a combination of:

-   -   pilocarpine, methimazole and at least one additional active        compound which is selected from baclofen, mifepristone, sorbitol        and naltrexone;    -   pilocarpine, methimazole and baclofen;    -   pilocarpine, methimazole and mifepristone;    -   pilocarpine, methimazole, mifepristone, sorbitol and baclofen;    -   pilocarpine, methimazole, mifepristone, sorbitol and rapamycin;    -   pilocarpine, methimazole, mifepristone, sorbitol and ketoprofen;    -   pilocarpine, methimazole, mifepristone, sorbitol and        flurbiprofen;    -   pilocarpine, methimazole, mifepristone, sorbitol, baclofen, and        naltrexone;    -   pilocarpine, methimazole, mifepristone, sorbitol, baclofen and        rapamycin;    -   pilocarpine, methimazole, mifepristone, sorbitol, naltrexone and        rapamycin;    -   methimazole and baclofen;    -   methimazole and cevimeline; or    -   pilocarpine and propylthiouracil.

The invention may be used for treating CMT or a related disorder in anymammalian subject, particularly human subjects. It is particularlysuited for treating CMT1a.

In this respect, a specific object of this invention is a method oftreating CMT1a in a subject, comprising administering to the subject aneffective amount of a combination of pilocarpine, or a prodrug thereof,and methimazole, or a prodrug.

A further object of this invention is a method of treating CMT1a, themethod comprising (1) assessing whether a subject has CMT1a and (2)treating the subject having CMT1a with an effective amount of acombination of pilocarpine, or a prodrug thereof, and methimazole, or aprodrug thereof. Determining whether a subject has CMT1a can be done byvarious tests known per se in the art, such as DNA assays.

LEGEND TO THE FIGURES

FIG. 1: Relative levels are represented as % of PMP22 mRNA expression inprimary rat Schwann cells treated for 24 hrs with compounds A and B. Onthe left is represented % of PMP22 mRNA level, 24 hrs after 1 mM or 1 μMof compound A (methimazole) addition. It is observed that PMP22 mRNA issignificantly decreased in primary Schwann cells, and that the lowerdose of compound A induces the most important PMP22 down-regulation. *:p<0.05; ***: p<0.001; significantly different from control (pairwisestudent t test). On the right, exposed 24 hrs to compound B(pilocarpine), PMP22 mRNA level of expression is significantlydown-regulated in primary Schwann cells even at very low doses (10 nMand 50 nM). Similarly, we observed that compound B (1 μM) significantlydecrease PMP22 protein level of expression after 24 hrs of incubation,by 38% in primary Schwann cells. This effect is still significant after48 hrs of incubation (−18%, p<0.001).

FIG. 2: Effect of selected drugs on PMP22 mRNA level of expressionquantified by RT-Q-PCR in RT4-D6P2T schwannoma cells. ***: p<0.0001:significantly different from control (=no drug). Bilateral Student's ttest on ΔΔCt values.

FIG. 3: Results of the motor assessment of the female rats in theBar-test throughout the treatment study presented in form of trends.WTplacebo: normal rats treated with placebo; TGplacebo: controltransgenic rats treated with placebo, TGptx25: transgenic rats treatedwith negative control substance, TGA: transgenic rats force fed withdaily dose of 0.2 mg/kg of methimazole; TGB: transgenic rats treatedwith daily dose of 0.35 mg/kg of pilocarpine.

FIG. 4: Electrophysiological assessment of the sensitive nerve potentialamplitude in CMT rats treated with drugs during 20 weeks. TGplacebo:control transgenic rats treated with placebo, TGptx25: transgenic ratstreated with negative control substance, TGA: transgenic rats treatedwith daily dose of 0.2 mg/kg of methimazole; TGB: transgenic ratstreated with daily dose of 0.35 mg/kg pilocarpine.

FIG. 5: The analysis of the post-mortem samples obtained from theanimals treated with drugs as described in the legend to FIG. 3. Therats treated with drugs and placebo during 20 weeks, were deeplyanesthetized and the entire sciatic nerves and soleus muscles werecarefully sampled and weighted. The graphs present the 30 mean values ofthese measurements.

FIG. 6: Positive effect of the Mix 1 in the gait evaluation test (femalerats); black line represents control rats treated with placebo; blackbold line represents transgenic rats treated with placebo; dotted linerepresents transgenic rats treated with Mix1. * p<0.05; black *: wtplacebo vs tg placebo; grey *: tg placebo vs tg mix1. Statistics arerealised with the Student bilateral test; mean is represented ±s.e.m.

FIG. 7: Positive effect of Mix2 on the excitability threshold of thecaudal nerve (white bars represent control male rats treated withplacebo; black bars represent transgenic male rats treated with placebo;grey bars represent transgenic male rats treated with Mix2. Statisticsare realised with the Student bilateral test; mean is represented±s.e.m.

FIG. 8: Positive effect of Mix2 after 4 weeks of treatment in the bartest (white bars represent control male rats treated with placebo; blackbars represent transgenic male rats treated with placebo; grey barsrepresent transgenic male rats treated with Mix2). Statistics arerealised with the Student bilateral test; mean is represented ±s.e.m.

FIG. 9: Positive effect on gait of Mix2 after 3 weeks of treatment(white bars represent the percentage of male rats in each group walkingwith a fluid gait; grey bars represent the not fluid gait; black barsrepresent the incapacity). Statistics are realised with the Studentbilateral test.

FIG. 10: Anti-allodynic effect in chronic OXALPN model. In blue areshown the reaction times in acetone test for control animals, in redoxaliplatin treated animals, in green color the animals treated withoxaliplatin and mix 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new therapeutic approaches for treatingCMT or related disorders. The invention discloses novel compositionsmade from combinations of known drugs and their new use for effectivecorrection of such diseases and may be used in any mammalian subject.

Within the context of the present invention, the term “CMT relateddisorder” designates other peripheral neuropathies both hereditary andacquired.

The major form of CMT, CMT1A, is caused by duplication of PMP22 gene.PMP22 is a major component of myelin expressed in the compact portion ofessentially all myelinated fibers in the peripheral nervous system.PMP22 protein interacts with another structural myelin protein P0, andtherefore, the altered PMP22/P0 protein ratio might influence thecompaction of myelin sheaths (Vallat et al., 1996; D'Urso et al., 1999).As demonstrated by in vitro studies, PMP22 protein is also involved inthe regulation of cell spreading in a Rho-dependent manner and thuscould affect axonal ensheathment (Brancolini et al., 1999). Moreover,PMP22 forms complexes with α6β4 integrins and could mediate theinteraction of Schwann cells with extracellular matrix (Amici et al.,2006; Amici et al., 2007). Furthermore, increased level of PMP22 proteincan alter the Arf6-regulated plasma membrane endosomal recycling pathwayand lead to accumulation of PMP22 in the late endosomes (Chies et al.,2003). It was also demonstrated that over expressed PMP22 proteinperturbs intracellular protein sorting and overloads the proteindegradation machinery in Schwann cells (Notterpek et al., 1997; Tobleret al., 2002; Fortun et al., 2003; Fortun et al., 2006; Fortun et al.,2007; Khajavi et al., 2007). Finally, PMP22 is directly involved in thecontrol of cell proliferation and programmed cell death (Sancho et al.,2001; Atanasoski et al., 2002) and mutant PMP22 protein was shown toprovoke profound reorganization and the aberrant expression of axonalion channels (Ulzheimer et al., 2004; Devaux & Scherer, 2005).

Consequently, the term “CMT related disorder” comprises peripheraldisorders such as ALS, toxic neuropathies, idiopathic neuropathies,diabetic neuropathy, cancer and HIV induced neuropathies, Guillain-Barrésyndrome.

In a preferred embodiment, CMT related disorder designates a neuropathy,such as demyelinating neuropathies, including HNPP (hereditaryneuropathy with liability to pressure palsies), CMT1B, CMT1C, CMT1D,CMT1X, CMT2A, CMT2B, CMT2D, CMT2E, CMT2-P0, severe demyelinatingneuropathies DSS (Dejerine-Sottas syndrome), CHN (congenitalhypomyelinating neuropathy), CMT4A, CMT4B1, CMT4B2, CMT4D, CMT4F, CMT4,AR-CMT2A, HSN1.

The invention is particularly suited for treating CMT1A.

As used herein, “treatment” of a disorder includes the therapy,prevention, prophylaxis, retardation or reduction of pain provoked bythe disorder. The term treatment includes in particular the control ofdisease progression and associated symptoms.

Also, the term compound designates the chemical compounds asspecifically named in the application, as well as any pharmaceuticallyacceptable salt, hydrate, ester, ether, isomers, racemates, conjugates,pro-drugs thereof.

Also, the term “combination” designates a treatment wherein at least twodrugs are co-administered to a subject to cause a biological effect. Ina combined therapy, the at least two drugs may be administered togetheror separately, at the same time or sequentially. Also, the at least twodrugs may be administered through different routes and protocols.

The invention shows that CMT or a CMT related disorder can be treated bya particular combination of drugs. More specifically, the inventionshows that methimazole and pilocarpine compounds, in combination(s), canbe used to treat CMT or related disorders. In this regard, theexperimental results show that a combination of methimazole andpilocarpine decreases significantly the level of RNA expression of PMP22gene in rat Schwann cells. Furthermore, PMP22 transgenic rats modellinghuman CMT disease have been treated daily with combinations containingmethimazole and pilocarpine. Significant improvement of behaviouralend-points was observed.

Pilocarpine:(3S,4R)-3-ethyl-4-[(3-methylimidazol-4-yl)methyl]oxolan-2-one

This drug, C₁₁H₁₆N₂O₂, has been approved for the treatment of i)symptoms of dry mouth from salivary gland hypofunction caused byradiotherapy for cancer of the head and neck; and ii) the treatment ofsymptoms of dry mouth in patients with Sjogren's syndrome.

Agonist of muscarinic receptors, it causes smooth muscle fiberscontraction (digestive tract, eye, bronchus), stimulates sudoral,salivary, bronchus and gastric secretions. Furthermore, it exhibits acomplex cardiovascular action, stimulating both parasympathomimetic(vasodilation) excitoganglionary pathways.

The inventors have demonstrated that pilocarpine, an agonist ofmuscarinic receptors, decreases expression of the PMP22 protein inSchwann cells in vitro. We propose that stimulation of muscarinicreceptors by pilocarpine leads, —likely, through complex set ofmolecular mechanisms, —to shifting in intracellular balance of Erk/Aktactivities, which regulate expression of myelin-associated proteinmarkers in opposite manner, to more pronounced Erk signalling (Ogata etal., 2004).

Without being bound by theory, it is postulated that stimulation ofmuscarinic receptors can modify activity of Akt and Erk kinases byseveral concomitant mechanisms (Ma et al., 2004; Anger et al., 2007).For instance, muscarinic receptors can selectively block signalling byIGF-1, but not PDGF receptors, by promoting inhibitory tyrosinedephosphorylation or inhibitory serine phosphorylation of IRS-1 by PKC.Then, muscarinic receptors could mediate intracellular transactivationof ERK signalling through Src-like Fyn kinase; as well, by decreasingactivity of adenylate cyclase, muscarinic receptors might be involved infunctional regulation of the Akt/Gsk-3β and Erk kinases also throughPKA-mediated mechanism. Finally, it was demonstrated that stimulation ofmuscarinic receptors can—through activation of AMPK—transiently causedephosphorylation of Akt, and thus decrease intracellular pool ofβ-catenin (Batty et al., 2004; King et al., 2006).

As a result, other muscarinic receptor agonists could be used as well,such as: Cevimeline (CAS number 107233-08-9), Carbachol (CAS number51-83-2), Methacholine (CAS numbers 55-92-5) and Bethanechol (CAS number674-38-4).

Methimazole: 1-methyl-3H-imidazole-2-thione

Methimazole inhibits the production of new thyroid hormones by blockingactivity of thyroid peroxidise, converting iodide to iodine andcatalyzing the incorporation of the resulting iodide molecule onto thephenol rings of tyrosines. Thus, methimazole can effectively decreasetranscriptional activity of thyroid hormone receptors. Additionally,methimazole has been reported to suppress prostaglandin production byattenuating prostaglandin H synthase activity (Zelman et al., 1984).Prostaglandins—through their cognate GPCR receptors—could furtheraugment activity of Akt signalling pathway, which promotes expression ofmyelin-related proteins (Ogata et al., 2004; Castellone et al., 2006).Accordingly, compounds which both inhibit thyroid hormone synthesis andaffect prostaglandin production or signalling are particularlyadvantageous for use in the present invention.

Other compounds related by action mode to methimazole are carbimazole(prodrug of methimazole—CAS number 22232-54-8) and propylthiouracil (CASnumber 51-52-5), and amiodarone (CAS number 1951-25-3).

As disclosed in the examples, compounds methimazole and pilocarpineexert a combined action leading to improved therapeutic effect againstCMT, permitting downscaling effective therapeutic doses with diminishedsecondary effects.

A particular embodiment of the invention resides in a combinationtherapy for treating CMT or a related disorder, particularly CMT1A,wherein said combination therapy comprises methimazole and pilocarpinecompounds and at least a third compound able to enhance the activity ofthis combination.

Another particular embodiment of the invention resides in a combinationtherapy for treating ALS, wherein said combination therapy comprisesmethimazole and pilocarpine compounds and at least a third compound ableto enhance the activity of this combination.

Another particular embodiment of the invention resides in a combinationtherapy for treating toxic neuropathy, wherein said combination therapycomprises methimazole and pilocarpine compounds and at least a thirdcompound able to enhance the activity of this combination.

A particular embodiment of the invention resides in a therapy fortreating CMT or a related disorder, wherein compounds methimazole andpilocarpine are used alone.

Another particular embodiment of the invention resides in a therapy fortreating CMT or a related disorder, wherein methimazole and/orpilocarpine are used in combination with at least one additional activecompound. In a preferred embodiment, the at least one additionalcompound is selected from the group of compounds listed in Table 1.

Another particular embodiment of the invention resides in a product orcomposition, which comprises any drug combination as disclosed in Table1.

Another particular embodiment of the invention resides in a combinationtherapy for treating CMT or a related disorder, comprising any drugcombination as disclosed in Table 1.

Another particular embodiment of the invention resides in a combinationtherapy for treating ALS, comprising any drug combination as disclosedin Table 1.

Another particular embodiment of the invention resides in a combinationtherapy for treating toxic neuropathy, comprising any drug combinationas disclosed in Table 1.

Therapy according to the invention is performed as drug combination,optionally in conjunction with any other therapy. It may be provided athome, the doctor's office, a clinic, a hospital's outpatient department,or a hospital, so that the doctor can observe the therapy's effectsclosely and make any adjustments that are needed.

The duration of the therapy depends on the stage of the disease, the ageand condition of the patient, and how the patient responds to thetreatment.

Additionally, a person having a greater risk of developing an additionalneuropathic disorder (e.g., a person who is genetically predisposed toor has, for example, diabetes, or is being under treatment for anoncological condition, etc.) may receive prophylactic treatment toalleviate or to delay eventual neuropathic response.

The dosage, frequency and mode of administration of each component ofthe combination can be controlled independently. For example, one drugmay be administered orally while the second drug may be administeredintramuscularly. Combination therapy may be given in on-and-off cyclesthat include rest periods so that the patient's body has a chance torecovery from any as yet unforeseen side-effects. The drugs may also beformulated together such that one administration delivers both drugs.

Formulation of Pharmaceutical Compositions

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to correct the effect ofelevated expression of PMP22 upon reaching the peripheral nerves.

While it is possible for the active ingredients of the combination to beadministered as the pure chemical, it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulations. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp using the formulation most suitably for the treatment. Suchmeasures make a patient pack specifically suitable for and adapted foruse for treatment with the combination of the present invention.

The drug may be contained in any appropriate amount in any suitablecarrier substance, and is may be present in an amount of 1-99% by weightof the total weight of the composition. The composition may be providedin a dosage form that is suitable for the oral, parenteral (e.g.,intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal,inhalant, skin (patch), or ocular administration route. Thus, thecomposition may be in the form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overan extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time; (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively, constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug, either alone or incombination, has (i) a narrow therapeutic index (i.e., the differencebetween the plasma concentration leading to harmful side effects ortoxic reactions and the plasma concentration leading to a therapeuticeffect is small; in general, the therapeutic index, TI, is defined asthe ratio of median lethal dose (LD50) to median effective dose (ED50));(ii) a narrow absorption window in the gastrointestinal tract; or (iii)a very short biological half-life so that frequent dosing during a dayis required in order to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner (single or multiple unit tabletor capsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, microcrystalline cellulose, starches includingpotato starch, calcium carbonate, sodium chloride, calcium phosphate,calcium sulfate, or sodium phosphate); granulating and disintegratingagents (e.g., cellulose derivatives including microcrystallinecellulose, starches including potato starch, croscarmellose sodium,alginates, or alginic acid); binding agents (e.g., acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and anti adhesives (e.g., stearic acid, silicas, or talc).Other pharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology.

The two drugs may be mixed together in the tablet, or may bepartitioned. For example, the first drug is contained on the inside ofthe tablet, and the second drug is on the outside, such that asubstantial portion of the second drug is released prior to the releaseof the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin, or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated metylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally byinjection, infusion or implantation (intravenous, intramuscular,subcutaneous, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well known to those skilled in theart of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in theform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamnine). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(glycolic acid) orpoly(ortho esters)).

Rectal Compositions

For rectal application, suitable dosage forms for a composition includesuppositories (emulsion or suspension type), and rectal gelatin capsules(solutions or suspensions). In a typical suppository formulation, theactive drug(s) are combined with an appropriate pharmaceuticallyacceptable suppository base such as cocoa butter, esterified fattyacids, glycerinated gelatin, and various water-soluble or dispersiblebases like polyethylene glycols. Various additives, enhancers, orsurfactants may be incorporated.

Percutaneous and Topical Compositions

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The emulsifying agents may be naturally occurring gums (e.g., gum acaciaor gum tragacanth)

The preservatives, humectants, penetration enhancers may be parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride,glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor alternatively plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. In a preferred embodiment,the drugs are formulated together, in the same excipient or carrier. Ifthere is sequential administration, the delay in administering thesecond (or additional) active ingredient should not be such as to losethe benefit of the efficacious effect of the combination of the activeingredients. A minimum requirement for a combination according to thisdescription is that the combination should be intended for combined usewith the benefit of the efficacious effect of the combination of theactive ingredients. The intended use of a combination can be inferred byfacilities, provisions, adaptations and/or other means to help using thecombination according to the invention.

Therapeutically effective amounts of two or more drugs that are subjectsof this invention can be used together for the preparation of amedicament useful for reducing the effect of increased expression ofPMP22 gene, preventing or reducing the risk of developing CMT1A disease,halting or slowing the progression of CMT1A disease once it has becomeclinically manifest, and preventing or reducing the risk of a first orsubsequent occurrence of an neuropathic event. Although the active drugsof the present invention may be administered in divided doses, forexample two or three times daily, a single daily dose of each drug inthe combination is preferred, with a single daily dose of all drugs in asingle pharmaceutical composition (unit dosage form) being mostpreferred.

Administration can be one to several times daily for several days toseveral years, and may even be for the life of the patient. Chronic orat least periodically repeated long-term administration will beindicated in most cases.

-   -   The term “unit dosage form” refers to physically discrete units        (such as capsules, tablets, or loaded syringe cylinders)        suitable as unitary dosages for human subjects, each unit        containing a predetermined quantity of active material or        materials calculated to produce the desired therapeutic effect,        in association with the required pharmaceutical carrier.

The amount of each drug in the combination preferred for a unit dosagewill depend upon several factors including the administration method,the body weight and the age of the patient, the severity of theneuropathic damage caused by CMT1A disease or risk of potential sideeffects considering the general health status of the person to betreated.

Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular patient may affect the dosage used.

Except when responding to especially impairing CMT disease cases whenhigher dosages may be required, or when treating children when lowerdosages should be chosen, the preferred dosage of each drug in thecombination will usually lie within the range of doses not above theusually prescribed for long-term maintenance treatment or proven to besafe in the large phase 3 clinical studies.

For example,

-   -   for methimazole from about 0.5 to about 15 mg per day if taken        orally. The special doses should be chosen if administered        topically.    -   for pilocarpine from about 0.1 to about 20 mg per day if day if        taken orally.

The most preferred dosage will correspond to amounts from 1% up to 10%of those usually prescribed for long-term maintenance treatment.

It will be understood that the amount of the drug actually administeredwill be determined by a physician, in the light of the relevantcircumstances including the condition or conditions to be treated, theexact composition to be administered, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thechosen route of administration. Therefore, the above dosage ranges areintended to provide general guidance and support for the teachingsherein, but are not intended to limit the scope of the invention.

Therapeutic Schema, Dosages and Routes of Administration

Below, the dosages for drug combinations (that differ in administrationroutes) in humans are described.

Methimazole and Pilocarpine

-   -   1—Administered orally as a single pharmaceutical composition:        methimazole from about 0.5 to about 15 mg and pilocarpine from        about 0.1 to about 20 mg every day orally for several months,        the most preferred dosages for both drugs in the composition        ranging from 0.6 to 35 mg per unit (per day).    -   2—Administered concomitantly orally for several months:        methimazole from about 0.1 to about 15 mg and pilocarpine from        about 0.02 to about 20 mg every day orally for several months,        the most preferred dosages for both drugs in the composition        ranging from 1.02 to 35 mg per unit (per day).    -   3—Administered concomitantly for several months: methimazole        from about 0.05 to about 15 mg and pilocarpine from about 0.01        to about 20 mg every day orally for several months, the most        preferred dosages for both drugs in the composition ranging from        0.06 to 35 mg per unit (per day).

The dosages of this drug in any combination among those disclosed in thepresent invention may differ in the formulations proposed for treatmentof men or women.

Additional aspects and advantages of the present invention will bedisclosed in the following experimental section, which should beconsidered as illustrative only.

Examples I. In Vitro Experiments Commercialized Rat Primary SchwannCells

Vials of rat Schwann cells (SC) primary culture (Sciencell # R1700) aredefrost and seeded at the density of 10 000 cells/cm2 in “SciencellSchwann cell medium” (basal medium from Sciencell # R1701) inpoly-L-lysine pre-coated 75 cm² flasks. The culture medium is composedof basal medium, 5% Fetal Bovine Serum (3H-Biomedical AB #1701-0025), 1%Schwann cell growth supplement (3H Biomedical AB #1701-1752), 1%Gentamicin (Sigma #G1397) and 10 μM of Forskolin (Sigma # F6886) topromote their proliferation.

After reaching confluency (4 to 10 days depending on cell batch),Schwann cells are purified by gentle agitation or by thy1.1immunopanning that allow SC isolation from adherent fibroblasts, toproduce cultures that are at least 95% pure. SC are then counted(Tryptan blue method) and seeded in poly-L-lysine pre-coated 75 cm²flask in the same SC medium. At confluency, cells are rinsed,trypsinized (trypsin-EDTA 1× diluted from Invitrogen #1540054), dilutedin PBS without calcium and magnesium) counted and platted in 12well-dishes (140 000 cells/well) in Sciencell Schwann cell medium with5% of FBS, 1% of cell growth supplement (CGS), 40 μg/ml of gentamicinand 4 μM Forskolin.

Custom-Made Rat Primary Schwann Cells

Primary Schwann cell cultures (SC) are established from Sprague-Dawleynewborn rats (between P0 and P2) sciatic nerves. All newborn rats aresacrificed and isolated in a Petri dish. Dissection is performed understerile conditions.

The dorsal skin is removed from the hind paw and the lower torso. Thesciatic nerve is isolated and transferred to a culture dish containingice-cold Leibovitz (L15, Invitogen #11415) supplemented with 1%penicillin/streptomycin solution (50 UI/ml and 50 μg/ml, respectively;Invitrogen #15070) and 1% of bovine serum albumin (BSA, Sigma A6003).Both nerves per rats are transferred in a 15 ml tube containing ice-coldL15. The L15 medium is then removed and replaced by 2.4 ml of DMEM(Invitrogen #21969035) with 10 mg/ml of collagenase (Sigma #A6003).Nerves are incubated in this medium for 30 minutes at 37° C. The mediumis then removed and both nerves are dissociated by trypsin (10% trypsinEDTA 10×, Invitrogen #15400054) diluted in PBS without calcium andmagnesium (Invitrogen #2007-03) for 20 min at 37° C. The reaction isstopped by addition of DMEM containing DNase I grade II (0.1 mg/ml Rochediagnostic #104159) and foetal calf serum (FCS 10%, Invitrogen #10270).The cell suspension was triturated with a 10 ml pipette and passedthrough a filter in a 50 ml tube (Swinnex 13 mm filter units, Millipore,with 20 μm nylon-mesh filters, Fisher). The cell suspension iscentrifuged at 350 g for 10 min at room temperature (RT) and the pelletsare suspended in DMEM with 10% FCS and 1% penicillin/streptomycin. Cellsare counted (Tryptan blue method) and seeded in Falcon 100 mm Primariatissue culture plates at the density of 5·10⁵ to 10⁶ cells/dish.

After one day of culture, the medium is changed with DMEM, 10% FCS, 1%penicillin/streptomycin and 10 μM of cytosine b-D-arabinofuranoside(Sigma #C1768). 48 hrs later, medium is eliminated and cells are washedthree times with DMEM. The SC growth medium is then added, composed ofDMEM, 10% FCS, 1% penicillin/streptomycin, 2 μM of Forskolin (Sigma#F6886), 10 μg/ml of bovine pituitary extract (PEX, Invitrogen #13028).The medium is replaced every 2-3 days.

After 8 days of culture (4 to 10 days depending on cell batches),Schwann cells reach confluency and the culture, containing a largeamount of contaminating fibroblasts, is purified by the thy1.1immunopanning method. After this purification, cells are suspended ingrowth medium at 10 000 cells/cm2 in poly-L-lysine pre-coated 75 cm²flasks. Once they reach confluency, cells are rinsed, trypsinized(trypsin-EDTA), counted and platted in 12 well-dishes (100 000cells/well).

Drug Incubation:

After cells being plated in 12 well-dishes, the medium is replaced by adefined medium consisting in a mix of DMEM-F12 (Invitrogen #21331020)complemented by 1% of N2 supplement (Invitrogen #17502), 1% L-Glutamine(Invitrogen #25030024) 2.5% FBS (Sciencell #0025), 0.02 μg/ml ofcorticosterone (Sigma # C2505), 4 μM Forskolin and 50 μg/ml ofgentamycin. Growth factors are not added to this medium, to promote SCdifferentiation.

24 hours later, the medium is replaced by a defined medium (DMEM-F12)complemented with 1% Insulin-Transferrin-Selenium—X (ITS, Invitrogen#51300), 16 μg/ml of Putrescine (Sigma # P5780), 0.02 μg/ml ofcorticosterone and 50 μg/ml of gentamicin. At this step, neitherprogesterone nor forskolin are present in the medium.

One day later, primary Schwann cells are stimulated by drugs during 24hrs (3 wells/condition). The preparation of each compound is performedjust prior to its addition to the cell culture medium.

Pilocarpine (SIGMA) was tested in primary Schwann cells at 10 μM to 10nM concentration range, while methimazole (SIGMA) was tested at 10 μM, 1μM, 100 nM and 10 nM.

Drugs are added to a defined medium composed of DMEM-F12, with 1%Insulin-Transferrin-Selenium—X (ITS, Invitrogen #51300), 16 μg/ml ofPutrescine, 0.02 μg/ml of corticosterone, 10 nM Progesterone and 50μg/ml of gentamicin. The absence of Forskolin before and during drugstimulation avoids adenylate cyclase saturation.

Cultured Schwannoma Cells

Rat schwannoma RT4-D6-P2T cell line (ATCC #CRL2468™) is defrosted inDMEM (ATCC #30-2002) and 10% FCS (invitrogen #10106). The cells aremaintained at 37° C. in a humidified incubator, in an atmosphere of air(95%.)—CO2 (5%). At passage n° 4, cells are dissociated bytrypsinization (+1 ml of Trypsin-EDTA, 0.25%-0.53 mM; Invitrogen) 5 to15 min at 37° C. The reaction is stopped by addition of DMEM containing10% of foetal bovine serum (FBS). Wells are counted in a Neubauercytometer using the trypan blue exclusion test (Sigma). The suspensionis triturated with a 10 ml pipette and the cells are then centrifuged at350×g for 10 min at room temperature. The pellet of dissociated cells isresuspended and seeded on the basis of 30 000 cells/ml in 12well-plates. 48 hrs later, the medium is replaced by a medium withoutserum (DMEM). After 15 hrs, RT4-D6P2T wells are stimulated by drugsadded to the cell culture medium at the chosen concentration. Thepreparation of each individual drug or drugs combination is performedjust prior to its addition to the cell culture medium.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-RT-PCR)

Quantitative RT-PCR is used to compare the levels of PMP22 mRNA afterdrug stimulation, relative with housekeeping RPS9 mRNA in RT4-D6P2T cellline.

8 hrs after drug incubation, cells are rinsed with cold sterilized PBS,total RNAs from each cell sample are extracted and purified from SCusing the Qiagen RNeasy micro kit (Qiagen #74004). Nucleic acids arequantified by Nanodrop spectrophotometer using 1 μl of RNA sample. TheRNA integrity is determined through a BioAnalyzer (Agilent) apparatus.

RNAs are reverse-transcribed into cDNA according to standard protocol.cDNA templates for PCR amplification are synthesized from 100 ng oftotal RNA using SuperScript II reverse-transcriptase (Invitrogen#18064-014) for 60 min at 42° C. in the presence of oligo(dT), in afinal volume of 20 μl.

cDNAs are subjected to PCR amplification using the “LightCycler® 480”system (Roche Molecular Systems Inc.) Each cDNA are diluted five timesbefore being used for PCR amplification. 2 μl of this cDNAs enters thePCR reaction solution with 5 μl of Master mix kit (Roche #04-887301001)in a final volume of 10 μl. Preliminary experiments ensured thatquantitation was done in the exponential phase of the amplificationprocess for both sequences and that the reaction efficiency is similarbetween target and housekeeping genes.

Taqman chemistry was used to perform RT-Q-PCR analysis. PCR reaction isperformed by amplification of rat PMP22 (NM 017037) by using 500 nM ofeach primers (F and R) and 200 nM of probe Taq1 from Sigma-Aldrich.

The following PCR conditions are used: Denaturation 5 min at 95° C.followed by 10 sec at 95° C., 40 sec at 60° C. and 10 sec at 72° C. andlmin at 40° C. (Forty amplification cycles). The relative levels ofPMP22 gene expression are measured following the Ct method comparing thequantity of products generated from the target gene PMP22 and the PMP22expression analysis by flow cytometry (FACS) 8 hrs, 24 hrs and 48 hrsafter drugs incubation, supernatants of primary rat Schwann cells arerecovered, centrifuged and frozen. SC are detached with trypsin-EDTA. Assoon as the majority of cells are in suspension, the trypsin isneutralised using DMEM with 10% FCS.

Supernatants with cells are recovered and centrifuged. The pellets ofcells are transferred in micro tubes, washed in PBS once and fixed witha specific solution (AbCys #Reagent A BUF09B). 10 minutes later, cellsare rinsed once with PBS and kept at 4° C.

Five days after cell fixation, all cell preparations with differentincubation times are labelled using the following protocol.

Cells are centrifuged at 7000 rpm for 5 minutes and the pellets aresuspended in a solution of permeabilization (AbCys #Reagent B BUF09B)and labelled with primary PMP22 antibody (Abcam #ab61220, 1/50) for 1 hrroom at temperature. Cells are then centrifuged at 7000 rpm for 5minutes and cell pellets are rinsed once in PBS. A secondary antibody isadded, coupled to Alexa Fluor 488 (goat anti-rabbit IgG, MolecularProbes #A11008, 1/100), for one hour at room temperature. Cells are thencentrifuged at 7000 rpm for 5 minutes and cell pellets are rinsed oncein PBS. The labelling is increased adding a tertiary antibody coupled toAlexa Fluor 488 (chicken anti-goat IgG, Molecular Probes #A21467, 1/100)for one hour incubation, at room temperature. Cells are then rinsed oncein PBS. Control without any antibody (unlabelled cells) is performed todetermine the level of autofluorescence and adapted the sensitivity ofthe photomultiplicators. Control with both secondary and tertiaryantibodies but without primary antibody, is performed to assess nonspecific binding of antibodies.

Data acquisition and analysis are performed with a FACS Array cytometerand FACS Array software (Becton Dickinson) on 5000 cells. ForwardScatter (FSC) correlated with cell volume (size) and Side Scatter (SSC)depending on inner complexity of cells (granularity) are analysed. Forexpression of PMP22, analysis is performed within the total cells andpercent of positive cells is calculated. Positive cells are cells withfluorescence intensity higher than the control with secondary antibody.

In order to quantify the number of SC, cells in control medium areanalysed using antibodies anti-S100 Protein.

Cells are prepared according to the following protocol: Schwann cellsare stained with antibody anti-S100 Protein (Dako #S0311, 1/100) for 1hr room at temperature. This antibody is labelled according to protocoldescribed above for PMP22 immunostaining but without incubation withtertiary antibody.

Results

We observed that PMP22 mRNA levels (FIG. 1) are significantly decreasedin primary Schwann cells, and that 1 μM dose of pilocarpine induces themost important PMP22 down-regulation. *: p<0.05; ***: p<0.001;significantly different from control (pairwise student t test). On theright, exposed 24 hrs to pilocarpine, PMP22 mRNA level of expression issignificantly down-regulated in primary Schwann cells even at low doses(10 nM and 50 nM). Similarly, we observed that pilocarpine (l μM)significantly decreases PMP22 protein level of expression after 24 hrsof incubation, by 38% in primary Schwann cells. This effect is stillsignificant after 48 hrs of incubation (−18%, p<0.001).

In another experiment drugs (listed in Table 1) are incubated with ratschwannoma cell line during 8 hrs and PMP22 mRNA expression level wasquantified by RT-Q-PCR.

The inventors have observed (FIG. 2) that while Methimazole (0.1 nm) andPilocarpine (0.001 nm), used as individual drugs, exert no significantactivity on PMP22 mRNA level of expression, this last one issignificantly decreased by their combination. The synergic activity ofthese two drugs is illustrated in FIG. 2. These data demonstrate that atchosen concentrations the combination of pilocarpine and methimazole isable to down regulate significantly the expression of PMP22 gene incultured Schwannoma cells, while pilocarpine and methimazole that areactive at higher concentrations in primary Schwann cells do not decreasethe level of expression of this gene in this system.

II. Experiments In Vivo in CMT Animal Model

The inventors have tested compounds and combinations for therapeuticeffect in CMT transgenic rat model, a hemizygous PMP22 transgenic ratsbearing three additional copies of mouse PMP22 gene show signs ofdemyelination in peripheral and cranial nerves, (Sereda et al., 1996;Grandis et al., 2004). At the mRNA level, an average 1.6-foldoverexpression of PMP22 in CMT rats correlates with the clinicalphenotype. A putative threshold level of PMP22 overexpression at whichthe wild type gene turns into a disease gene represents an obvious“target” to reach by treatment aimed at reducing PMP22 gene expression.

This CMT rat model is a good approximation of human CMT1A disease from aclinical point of view. Adult CMT rats exhibit a slowing of motor nerveconduction velocity with values similar to those of CMT1A patients,i.e., less than 50%. After sciatic nerve stimulation, compound muscleaction potentials show reduced amplitudes and desynchronization. Thehistological and electrophysiological changes precede the overt clinicalsigns of motor impairment (Sereda et al., 1996, 2003). Axonal loss,confirmed by histological pronounced muscle atrophy, matches the humanCMT1A symptoms.

The CMT rats already served as a model for an experimental CMT1A therapy(Meyer zu Hörste et al. 2007). In this model of CMT1A disease, the overtand hidden signs of pathology (locomotor deficiency, particularalterations in electrophysiological and tissue characteristics and,finally, the level of PMP22 over-expression appear to be the closest tothose found in CMT1A patients).

The inventors have tested the compounds and combinations for therapeuticeffect in a rat model. The experimental groups are formed with youngrats of both genders separately. The rats are assigned to the groupsfollowing randomization schedule based on the body weight. In someexperiments the randomization is based on the performances of the ratsin the bar test. Both genders are represented by separate controllittermates groups that are numerically equal or bigger than thetreatment groups.

The rats are treated chronically with drugs—force fed or injected byAlzet osmotic subcutaneous pump (DURECT Corporation Cupertino, Calif.),depending on each drug bioavailability during 3 or 6 weeks.

The animals are weighted twice a week in order to adjust the doses togrowing body weight. If the osmotic pump is chosen for the treatmentadministration, the doses of the drug are calculated on the basis of theestimated mean body weight of the animals expected for their age overthe period of the pump duration (6 weeks). The pumps are re-implanted ifnecessary, with the appropriated anaesthesia protocol.

Behavioural Tests

Each three or four weeks the animals are subjected to a behaviouraltest. Each test is conducted by the same investigator in the same roomand at the same time of the day; this homogeneity is maintainedthroughout entire experiment. All treatments and genotype determinationare blinded for the investigator. “Bar test” and “Grip strength” hasbeen mainly used to access the performance throughout study. Theschedule of the bar test may change as the animal growth (in order toavoid the bias due to the learning, for example).

The assay of the grip strength allows detection of subtle differences inthe grip performance that seems to be composed of the muscle force,sensitivity status (for instance, painful tactile feelings may changemeasured values of the force), behavioural component (“motivation”). Thevalues differ between fore and hind limbs and greatly depend on the ageof the animals.

The grip strength test measures the strength with which an animal holdson to a grip with its forepaws or its hindpaws separately. A dynamometeris placed with a grip to measure the strength (Force Gauge FG-5000A).The rat is held by the experimenter in a way that it grasps the gripeither with its forepaws or with its hind paws and pulls gently the ratbackwards until it releases the grip. The force measured when the animalreleases the grip is recorded.

Two successive trials measuring the forepaws and two successive trialsmeasuring the hindpaws strength per animal are processed; only themaximum score (one for forepaws and one for hindpaws) is noticed (in N).

The Bar Test

The bar test evaluates rats' ability to hold on a fix rod. Pmp22 ratswhich display muscular weakness, exhibit a performance deficit in thistest (Sereda et al, 1996). The rat is placed on its four paws on themiddle of the rod (diameter: 2.5 cm; length: 50 cm; 30 cm above thetable). Trials are performed consecutively; the number (5 or 10) and theduration (30 or 60 sec) of trials in our experiments have been dependingon batches of the animals. This variability in the testing has beenintroduced in order to determine the schedule appropriated to the bestdetection of the motor deficiency in the CMT rats in the course of theexperiments.

Performance indices are recorded on each session:

-   -   The number of trials needed to hold for 60 sec or 30 sec on the        rod.    -   The mean time spent on the bar (i.e. the fall latency) in each        trial and the average on the session. In the experimental        procedures the session ends after the rat has stayed two times        for a cut-off time, i.e. 30 or 60 s, on the bar. A performance        of the cut-off time (30 s or 60 s) is assigned to trials not        completed.    -   The number of falls.

General Health Assessment

Body weights, overt signs (coat appearance, body posture, tremor) of theanimals are monitored throughout the experiment. The rating scale isused for recording: 0=normal, 1=abnormal.

The Gait

Each rat is observed in a novel rat cage (dimensions 55×33×18 cm)without litter for five minutes. The gait of rats is evaluated with 4parameters:

-   -   Score 0: normal gait (fluid)    -   Score 1: abnormal gait (not fluid or the rat has a slight limp)    -   Score 2: moderate incapacity (the rat drags one's leg and is        able to put it right and walk)    -   Score 3: serious incapacity (the rat drags its one's or both        hindpaws but is unable to put it/them right).

Inclined Plane Test

The sliding apparatus had a 30×50 cm plexiglas plane that could beinclined at an angle of 0° (horizontal) to 60°. Each rat was initiallyplaced on the 25°-angled inclined plane in the up-headed position(head-up orientation); two trials separated by 1 min are performed. 30min later, the same experiment is realized on a 35°-angled inclinedplane then on 40°-angled inclined plane. During this time the rat wasreturned to its cage. The plane is cleaned after each trial.

The performances of rats are evaluated by 4 different scores:

-   -   Score 0: no slide    -   Score 1: a little slide (one or two paws)    -   Score 2: a moderate slide (4 paws) but not until the end of the        plane    -   Score 3: the rat is sliding until the very bottom of the plane.

Electrophysiology

When appropriate, the rats are subjected to electrophysiologicalevaluation: the sensitive nerve conduction velocity as well as latenciesand potential amplitude are measured.

NCV measurement and potential acquisition (subcutaneous) were performedwith the help of the chain composed of an amplificator (AM System 1700and/or EMG-UTC), a stimulator (Havard apparatus 223) and a computerequipped with an acquisition card and a software for acquisition(SPATOL) and for the signal treatment (CALVISE).

Animals were anesthetized using ketamine/xalazine and maintained on thethermostated plate at 37° C. throughout the test (anaesthetics weresupplemented as needed). Stimulating silver needle electrodes wereinserted in the proximal part of the tail. The recording electrode wasinserted subcutaneously through about 1 mm of skin in the distal part ofthe tail (4 and 6 cm from the stimulating electrode). Constant-currentsquare-wave stimuli, 0.2 s in duration, were administered at a frequencyof 0.3 per second. Responses, amplified 5.000-20.000, were visualizedand collected on a computer based data acquisition system. Latencieswere measured at each wave onset (defined as the first clearlyidentifiable deflection from the baseline). Peak to peak amplitudes ofthe largest deflections were calculated to determine maximum amplitude.For each recording, measurements were performed on the averagedresponses to at least ten identical stimuli.

Sensory related conduction velocity (SNCV) was calculated by division ofthe distance between the stimulating cathodes by the difference betweenthe correspondent latencies obtained from the two sites of stimulation.

Histological Measurements

Upon the final tests (treatments being continued until the last day),the rats were euthanized. Hind feet of wild-type and transgenic ratswere dissected and fixed by immersion in formal 4% solution for 48 hrs,and transferred in a 10% formol solution for 2 additional days. Afterbeing rinsed 15 min in water, they were then processed fordecalcification for 26 hrs (Labonord Décalcifiant rapide No. 3#DC3_(—)09128300).

Feet were then transversally sectioned in two pieces that are processedfor osmium coloration.

Tissues are hang up above 1% osmium tetroxyde solution (VWR, Osmium VIIIoxide 0.5 g #20551.076) for 24 hrs and then rinsed with demineralisedwater for 4 to 6 hrs. Tissues are then dehydrated in automate ofembedding (VIP2000 Vertical, Bayer Diagnostics) and classically embeddedin paraffin.

Tissue sections of 4 μm are dehydrated in successive xylene and alcoholbaths and mounted (Pertex glue, #T/00811_MICROM) for further analysis.

Image Analysis

Sections from the 6 animals were carefully chosen to illustrate the sameanatomical level (toe junction) and are analyzed under Olympusmicroscope coupled with Saisam microvision software (Archimed Pro®1997-2000 by Microvision Instruments). Peripheral nerve is localized andanalyzed as follows: circulary myelinated fibers are counted (in abundle nerve at least 150 fibers/animal). Cylindraxe diameterscorresponding to inner perimeters of myelinated fibers and outerperimeters of the same fibers are determined. Then, we compared thedistribution of myelin thickness and axons diameter in wild type andtransgenic animals.

Furthermore, we measure optical density of the same nerve section todetermine whether the non myelinated fibers content (not visible onosmium sections and so not analyzed) is higher in transgenic animals(reflected by a global pale appearance of the nerve section).

Finally, all foot muscles, present on the same section than used fornerve analysis, are manually outlined to determine global musclesurface. We then calculated the ration “muscle content/section surface”.

Sciatic nerves are excised and used for weighting as well as formolecular biology and/or biochemical essays. (RT-Q-PCR for PMP22 mRNAand Western blots for myelin proteins quantifications; Cayman's EIAkits—for biochemical markers such as arachidonic acid metabolites, HPLCquantification for steroids and amines, ELISA for CNTF, IL-6 etc.)performed according to generally used protocols and for analyticalprocedures (drug concentrations measures).

The hind limbs muscles (soleus) are sampled, weighted, snap-frozen andpreserved at −80° C. until analysis (the same as for sciatic nerves).

Results

Methimazole (0.35 mg/kg daily dose) and pilocarpine (0.2 mg/kg dailydose) administered by forced feeding improve bar test performancesthroughout the treatment procedure (FIG. 3), while compound PXT25 (whichis presented here only for the sake of comparison) hardly shows anyimprovement.

The motor performances were on average 3-fold less successful indifferent CMT TG rats treated with placebo compared with Wild type (WT)group. The treatment with methimazole and pilocarpine allowedimprovement of the TG animals in this experiment, the effect becomesstatistically significant as early as after 8 weeks of theforce-feeding.

The data show that CMT rats treated with methimazole and pilocarpine atthese relatively high doses became significantly more performingcompared with the placebo group. The group treated with compoundpilocarpine even recovered the level of performance which is no moresignificantly differs from that of the WT placebo group.

The SNAP measured on the distal portion of the tail was found to besignificantly diminished in the TG placebo group that may reflect theimportant axonal loss which in turn is due to the demyelination. Thiselectrophysiological parameter turns to be significantly improved uponthe treatment with compound A, (FIG. 4) while SNAP for the transgenicrats treated with compound B is approaching the nominal 5% threshold forsignificance.

This observation allows us to suppose that the action of methimazole mayprevent the axon loss, even if the myelination status of the peripheralnerves is not measurably improved. The effect of pilocarpine seems to beessentially the same, even if because of the intra-group variability thedifference with the placebo group parameter failed to reach statisticalsignificance. In CMT1A, (sensory nerve action potential (SNAP) amplitudewas more reduced and SNAP duration more prolonged than in CMT2. Thereduction of composed muscle action potential (CMAP) and SNAP amplitudesin CMT1A is probably a combined effect of demyelination and axonaldysfunction. (Bienfait et al., 2006).

At the end of the study morphometrical analysis has been performed. Themeasurement of the hindlimb tissues reveals that the sciatic nerves andsoleus muscles are significantly reduced in the CMT female rats treatedwith placebo compared with the control WT rats. (FIG. 5)

These deficiencies appear to be completely corrected by compound Atreatment: the absolute masses of the muscles and the nerves are evenhigher than in the control WT rats, while the entire body weight israther diminished in the compound A group comparatively with the placebogroup (data not shown). The effect of pilocarpine treatment on thehindlimb muscle and nerves appears to be smaller than that ofmethimazole.

The Mix1 (Table 1) at 50 times lower doses of methimazole (4 mkg/kg) andpilocarpine (7 mkg/kg) improves the gait score of female rats after 10weeks of forced fed treatment as shown in FIG. 6. We observed a positivetrend after 6 weeks of treatment.

The following figures illustrate the positive effect of the mix 2(Table 1) containing pilocarpine (7 mkg/kg); methimazole (4 mkg/kg),miferpristone (40 mkg/kg), naltrexone (4 mkg/kg; baclofen (60 mkg/kg andsorbitol (2 mg/kg) on male rats in 3 different behavioural andelectrophysiological tests.

The FIG. 7 reveals that the Mix2 at these doses decreases the raise ofthe excitability threshold, found in the CMT placebo rats, after a nervecaudal electrical stimulation.

The FIG. 8 shows the positive effect of the Mix2 on the males'performances on the bar test; after 4 weeks of treatment, the number offalls is decreased and the time spent on the rod is increased.

The figure illustrates the fact that the Mix2 improves also the gaitperformances of the male rats after 3 weeks of treatment; the percentagerats walking with a fluid gait is increased by 35% for the treated ratscompared to CMT placebo rats.

Similar results are produced for other combinations and summary ofresults could be shown in Table 1.

TABLE 1 POS: reversion of disease symptoms in vivo effect in vivo POSPOS POS POS POS POS POS POS POS POS combination mix1 mix2 mix3 mix4 mix5mix6 mix7 mix8 mix9 mix10 mifepristone * * * * * *pilocarpine * * * * * * * * * methimazole * * * * * * * * *sorbitol * * * * * * naltrexone * baclofen * * * rapamycin *ketoprofen * flurbiprofen * cevimeline * propylthiouracil * effect invivo POS POS POS POS POS POS POS POS combination mix11 mix12 mix13 mix14mix15 mix16 mix17 mix18 mifepristone * * * pilocarpine * * * * * * * *methimazole * * * * * * * * sorbitol * * * naltrexone * * baclofen *rapamycin * * * ketoprofen * flurbiprofen * cevimeline propylthiouracil

These data show that, in vivo, the combinations and regimens of thisinvention could allow effective treatment of CMT.

III. In Vivo Effect in a Model of Toxic Neuropathy

The drug treatments or regimen are orally administered from the daybefore the first intraperitoneal injection of Oxaliplatin 3 mg/kg (D−1)until the day before the last testing day

(D16). Animals belonging to the Oxaliplatin-treated group are doseddaily with distilled water (10 ml/kg). Animals are dosed with the testedtreatment and distilled water daily during the morning whereasOxaliplatin is administered on the afternoon.

During the testing days (i.e. D1, D4, D10), the treatment and distilledwater are administered after the test. Regarding the testing day (D4),including compounds and vehicle administrations and Oxaliplatininjection, the treatment and distilled water are administered prior tothe injection of Oxaliplatin after the test. Animals from thereference-treated group are dosed only during the testing days (i.e. D1,D4, D10 and D17).

Cold allodynia is assessed by measuring the responses to thermalnon-nociceptive stimulation (acetone test) on D1 (around 24 h after thefirst injection of Oxaliplatin 3 mg/kg (acute effect of Oxaliplatin), onD4, D10 and (chronic effect of Oxaliplatin) and on D17 (residual effectof Oxaliplatin one week after completion of treatment).

Testing is done using the acetone test 2 h post-administration of thereference.

The reference substance is Gabapentin, 100 mg/kg, per os (once a day×4testing days).

Acetone Test

Cold allodynia is assessed using the acetone test. In this test, latencyof hindpaw withdrawal is measured after application of a drop of acetoneto the plantar surface of both hindpaws (reaction time) and theintensity of the response is scored (cold score).

Reaction time to the cooling effect of acetone is measured within 20 sec(cut-off) after acetone application. Responses to acetone are alsograded to the following 4-point scale: 0 (no response); 1 (quickwithdrawal, flick of the paw); 2 (prolonged withdrawal or markedflicking of the paw); 3 (repeated flicking of the paw with licking orbiting).

Six trials by rat are performed. For each experimental group, theresults are expressed as the cumulative cold score defined as the sum ofthe 6 scores for each rat together ±SEM. The minimum score being 0 (noresponse to any of the 6 trials) and the maximum possible score being 18(repeated flicking and licking or biting of paws on each of the sixtrials).

Gabapentin: Source: Zhejiang Chiral Medicine Chemicals, China

Oxaliplatin: Source: Sigma, France

Results

Results of testing of mix2 in oxaliplatine are shown on FIG. 10. It isevident that mix 2 protects animals from neuropathy induced by toxicdrug treatment.

IV. In Vivo Effect in a Model of ALS Animal Model

We have chosen the SOD 1^(G93A) rat model (generated by Howland D S etal, 2002) to mimic the Amyotrophic Lateral Sclerosis pathology. Thismodel overexpresses the mutated SOD1 gene in spinal cord, many brainregions as well as peripheral tissues (Howland D S et al, 2002). Theonset of the motor neuron disease of this model is about at 115 days(Howland D S et al, 2002); it appears as hind limb abnormal gait. In fewdays, the paralysis of hind limb arises.

Experimental Procedures

We obtained colonies by crossing breeder SOD1^(G93A) rats with SpragueDawley female rats. Heterozygous SOD1^(G93A) rats were identified withpolymerase chain reaction (PCR) of tail DNA with primers specific forhSOD1 (Howland D S et al, 2002). Animals were maintained in a room withcontrolled illumination (lights on 0500-1900 h) and temperature (23±1°C.), and given free access to food and water. All the animal proceduresin the present study were carried out in accordance with the guidelinesstandards of animal care.

Body weight measurement was performed every week and behavioral testsbegan at an age of 60 days and continued until endpoint. The treatmentswere administered every day per oral or subcutaneous way from the age of5 weeks.

Observation test: characterization of the general aspect Each rat wasobserved in a novel rat cage (dimensions 55×33×18 cm) without litter forfive minutes. 5 different parameters are recorded:

The Gait

-   -   score 0: normal gait (fluid)    -   score 1: abnormal gait (not fluid or the rat has a slight limp)    -   score 2: moderate incapacity (the rat drags one's leg and is        able to put it right and walk)    -   score 3: serious incapacity (the rat drags its one's or both        hind paws but is unable to put it/them right)

The Coat Aspect

-   -   score 0: clean and silky coat    -   score 1: piloerection or dirty coat

The Tremor

-   -   score 0: no tremor    -   score 1: tremor

The Body Position

-   -   score 0: normal    -   score 1: abnormal (flattened or arching its back)

The Hindpaws Position

-   -   score 0: normal    -   score 1: spread hindpaws

The motor score test: characterization of the motor deficit

This test evaluates the ability of rats to right themselves within 30sec of being turned on either side (righting reflex) (Gale K. et al,1985).

A non-parametrical scoring system was used following these criteria(Matsumoto A. et al, 2006; Thonhoff J R et al, 2007):

-   -   score 0: the rat is unable to right itself from either side        within 30 sec;    -   score 1: the rat is unable to right itself from only one side        within 30 sec;    -   score 2: the rat is able to right itself from both sides within        30 sec but is unable to stand in the cage; it is always dragging        some parts of body;    -   score 3: the rat is able to right itself from both sides within        30 sec, is unable to stand in the cage but is not dragging some        parts of body;    -   score 4: the rat is able to right itself from both sides within        30 sec, is able to stand in the cage but has visible functional        deficits;    -   score 5: the rat is able to right itself from both sides within        30 sec, is able to stand in the cage and no visible functional        deficits.

The end-point of disease is fixed at score 0; the rat is theneuthanized.

Inclined plane test: characterization of the motor deficit

The sliding apparatus had a 30×50 cm plexiglas plane that could beinclined at an angle of 0° (horizontal) to 60°. Each rat was initiallyplaced on the 25°-angled inclined plane in the up-headed position(head-up orientation); two trials separated by 1 min are performed. 30min later, the same experiment is realized on a 35°-angled inclinedplane then on 40°-angled inclined plane. During this time the rat wasreturned to its cage. The plane is cleaned after each trial.

The performances of rats are evaluated by 4 different scores:

-   -   score 0: no slide    -   score 1: a little slide (one or two paws)    -   score 2: a moderate slide (4 paws) but not until the end of the        plane    -   score 3: the rat is sliding until the very bottom of the plane

The wire mesh test: characterization of the motor ability in difficultsituation

A wire mesh was placed in contact with a box at the top (at an angle of70°) and the edge of a table at the bottom (Thonhoff J R et al, 2007).Each rat was placed on the bottom of the wire mesh and motivated toascend by placing their littermates in the box at the top. Each rat wastrained once a week (3 trials).

The recorded parameter was the latency time to reach the top of the wiremesh.

The open field test: characterization of the locomotor activity

The locomotor activity was measured in a Plexiglas box (45×45×30 cm,Acti-Track by BIOSEB, Lyon, France) with 16 photo-cell beams followingthe two axes, 1 and 5 cm above the floor.

The spontaneous and exploratory activity of each rat was evaluatedduring 3 hours.

4 parameters are recorded (the total travelled distance, the number ofrearings, the percentage of travelled distance and of time spent in thecenter of the openfield).

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1. (canceled)
 2. A method of treating toxic neuropathy in a subject inneed thereof comprising the administration to said subject of acombination product or composition comprising: a) pilocarpine or a saltthereof; b) methimazole, carbamizole or a salt thereof; and c) apharmaceutically acceptable carrier or excipient.
 3. The method of claim2, wherein said combination product comprises: a) a salt of methimazoleand a salt of pilocarpine; b) a salt of methimazole and pilocarpine; c)methimazole and pilocarpine; d) methimazole and a salt of pilocarpine;e) carbamizole and pilocarpine; f) carbamizole and a salt ofpilocarpine; g) a salt of carbamizole and pilocarpine; or h) a salt ofcarbamizole and a salt of pilocarpine.
 4. The method of claim 2, whereinsaid combination product or composition further comprises at least oneadditional active compound.
 5. The method of claim 4, wherein said atleast one additional active compound is selected from baclofen,mifepristone, sorbitol, naltrexone, rapamycin, ketoprofen orflurbiprofen, or salts thereof.
 6. The method of claim 4, wherein saidcombination product or composition further comprises: a) baclofen; b)mifepristone; c) sorbitol; d) naltrexone; e) rapamycin; f) cevimeline;g) ketoprofen; h) flurbiprofen; i) mifepristone, sorbitol and baclofen;j) mifepristone, sorbitol and rapamycin; k) mifepristone, sorbitol andketoprofen; l) mifepristone, sorbitol and flurbiprofen; m) mifepristone,sorbitol, baclofen, and naltrexone; n) mifepristone, sorbitol, baclofenand rapamycin; or o) mifepristone, sorbitol, naltrexone and rapamycin;or salts thereof.
 7. The method of claim 6, wherein said combinationproduct or composition further comprises baclofen, mifepristone,sorbitol, and naltrexone, or salts thereof.
 8. The method of claim 1,wherein said subject is a human.
 9. The method of claim 1, wherein saidcombination product or composition is administered orally.
 10. Themethod of claim 1, wherein said combination product or composition isadministered repeatedly.